Immunization with live, replication-competent Ad-HIV or Ad-SIV recombinant vaccines primes strong antibody responses that develop following administration of booster immunizations with envelope protein. These antibodies display many functional activities. The most desirable for an HIV/AIDS vaccine is broadly neutralizing activity that can prevent infection following exposure to multiple clades or subtypes of the virus that circulate worldwide. However, neither this vaccine approach or others has been able to elicit such antibodies. More readily induced antibodies lack neutralizing activity but broadly react with multiple viral isolates and mediate effector functions via interaction with Fc receptors on a variety of cell types. HIV/SIV infection is initially manifested as small foci of infected cells. Within 2-6 days, virus spreads from these cell foci to draining lymph nodes, leading to systemic infection. The non-neutralizing functional antibody activities can help control the initial viral burden by limiting the spread of virus from the foci of infection. Our replicating Ad-recombinant prime/envelope boost vaccine regimen elicits such activities including antibody dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) and have associated them with vaccine-induced protective efficacy in SIV and chimeric SHIV Rhesus macaque models which appropriately mimic HIV infection in people. Recently we investigated another non-neutralizing activity, antibody-dependent complement mediated lysis (ADCML). We found that antibodies induced by our vaccine regimen mediated ADCML of both SIV virions and SIV-infected cells. Further, a modest correlation of ADCML lysis of SIV virions with reduced infection rate was seen in males but not females. This prompted us to look in greater depth at ADCML activity between the sexes. Gag-specific IgG and gp120-specific IgG and IgM correlated with SIV lysis in females while Env-specific IgM correlated with SIV-infected cell lysis in males indicating sex differences in vaccine-induced antibody characteristics and function. In fact, viral envelope-specific antibody functional correlates between ADCC, ADCP, and ADCML varied depending on the sex of the vaccinees. Additionally, glycosylation of antibody Fc regions differed between males and females and influenced corresponding functional activities. This latter result not only suggests mechanisms that can be employed to modify antibody functional activity, but also continues to indicate the importance of evaluating vaccine-induced responses in both sexes. We had previously shown a sex bias in response to SIV vaccination, with females but not males exhibiting delayed infection following repeated low-dose challenges to SIV. This previous outcome was associated with local mucosal immunity. The current result extends the mechanisms associated with the vaccine-induced sex bias to additional systemic immune responses. Overall, these data continue to suggest that sex influences vaccine-induced antibody function and should be considered in design of globally-effective HIV vaccines in the future. This is an important issue as approximately 50% percent of HIV infected people worldwide are women. We are continuing exploration of the sex bias by transcriptome analysis and analysis of the microbiome. We are studying individual cell types in order to further understand their contribution to overall vaccine-induced immunity. Neutrophils are the most prevalent leukocyte and exert considerable influence on the innate immune response, with increasing evidence that they also contribute substantially to adaptive immunity. Their innate functionality entails the release of a vast array of cytokines and chemokines. They are stimulated by various chemoattractants and subsequently traffic to sites of inflammation, where they can kill invading pathogens via phagocytosis, degranulation, or by releasing neutrophil extracellular traps (NETs). They contribute to adaptive immunity through immune cell crosstalk that can be both immunostimulatory and immunoregulatory, as well as by aiding in the resolution of inflammation. We have shown that neutrophils, together with antibodies elicited by our vaccine regimen, mediate ADCP. We found that rhesus macaque neutrophils can also provide help to B cells, a phenomenon previously reported in mice. B cells co-cultured with neutrophils were activated, underwent class switching, and produced antibodies. Mechanistically, this B-cell help was not aided by addition of IL-10 and was largely contact-dependent. Overall, immune stimulation of neutrophils by vaccination or antigenic exposure imparts a greater ability to contribute to the adaptive immune response. Harnessing this granulocytic response has the potential to improve vaccine efficacy. We have also studied B1 cells in naive and chronically SIV-infected macaques. B1 cells are the primary source of protective natural antibodies, and they can modulate T cell activation. T cells play a central role in defense against viral infections and this function is compromised when they become exhausted during chronic infection. B1 cells express PD-L2, a key factor involved in the induction of cell exhaustion, suggesting the cells might have immunosuppressive potential. We have explored whether B1 cells contribute to generation of exhausted T cells during chronic SIV infection. We found that a splenic B1 cell subset, CD11b+ B1 cells, correlated directly with higher plasma viral loads in chronically infected macaques, and also with the frequency of exhausted CD4+ T cells in males but not females. Expression levels of PD-L2 on these cells were directly associated with the frequency of the exhausted T cells. These observations further point to sex differences in immune cell function which may lead to differences in viremia control. The mechanism(s) by which B1 cells exert their suppressive potential is being further investigated. Additional interaction of B and T cells occurs in germinal centers of secondary lymphoid organs. T follicular helper (TFH) cells are fundamental in germinal center (GC) maturation and selection of antigen-specific B cells. While GC-resident TFH cells have been characterized in mice and humans, much less is known concerning their role in B cell maturation and development of humoral responses in the Rhesus macaque SIV model, the main preclinical model used in HIV vaccine development. We have comprehensively characterized GC TFH cells of Rhesus macaques over the course of vaccination to elucidate GC formation and SIV humoral response generation, studying lymph node biopsies in macaque subgroups pre-vaccination and at days 3, 7, or 14 after mucosal vector priming and systemic Envelope protein boosting. Evaluations of GC TFH and GC B cell dynamics and correlation analyses supported a significant role of early GC TFH cells in providing B cell help during initial phases of GC formation. GC TFH responses at day 3 post-prime were consistent with early generation of Env-specific memory B cells in GCs and elicitation of prolonged Env-specific humoral immunity in the mucosa. GC Env-specific memory B cell responses elicited early post-boost correlated significantly with decreased viremia post-infection. Our results highlighted the importance of early GC TFH cell responses for robust GC maturation and generation of long-lasting SIV specific humoral responses at mucosal and systemic sites. Finally, an on-going pre-clinical vaccine study in rhesus macaques is exploring induction of mucosal immunity elicited by our vaccine regimen, and whether exposure to non-infectious viral particles released mucosally as a result of microbicide treatment will boost the vaccine-induced responses. Such a microbicide-vaccine regimen would provide enhanced protection against HIV infection.